Botulinum neurotoxins with modified light chain specifity and methods for producing same

ABSTRACT

A protease directed to a non-neuronal SNARE protein is described. The protease is produced by selective mutation of a botulinum neurotoxin light chain, and is characterized utilizing a reporting construct that includes all or part of the non-neuronal SNARE protein. Such a protease has utility in the treatment of diseases associated with hypersecretion, where the hypersecretion is mediated by a non-neuronal SNARE protein.

This application is a divisional application of U.S. patent applicationSer. No. 14/824,986, filed Aug. 12, 2015, which claims priority to U.S.Provisional Application No. 62/036,412, filed Aug. 12, 2014 and U.S.Provisional Application No. 62/142,400, filed Apr. 2, 2015. These andall other referenced extrinsic materials are incorporated herein byreference in their entirety. Where a definition or use of a term in areference that is incorporated by reference is inconsistent or contraryto the definition of that term provided herein, the definition of thatterm provided herein is deemed to be controlling.

FIELD OF THE INVENTION

The field of the invention is botulinum neurotoxins.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Hyper-secretion from disease-specific cell types is characteristic ofmany endocrine, immune, and secretory diseases. For example, mast cellsecretion underpins anaphylaxis, allergic, autoimmune, and otherinflammatory diseases while mucin secretion from epithelial cellscontributes to cystic fibrosis (CF) and chronic obstructive pulmonarydisease (COPD). Reducing secretion by targeting the core machineryrequired for secretion can provide a new and effective treatmentmodality for such diseases. Secretion from such disease-specific celltypes is mediated by SNARE proteins, a family of membrane associatedproteins that form complexes which mediate vesicle fusion with theplasma membrane and subsequent release of vesicle contents.

Non-neuronal SNAREs are essential for endocrine and metabolic pathwaysthat regulate release of hormones, growth factors, and other signalingmolecules. Dysfunction in such secretion pathways results in disease.The non-neuronal Snare protein SNAP-23, for example, is essential forsecretion in multiple disease pathways, including IL-6 and TNF releasein arthritis, mucin hypersecretion in COPD, CF, and idiopathicbronchiectasis, platelet secretion in blood hemostasis, insulinsecretion in diabetes, renin release in blood pressure regulation, andmatrix-degrading enzyme release in tumor cell invasion. Similarly thenon-neuronal SNARE protein SNAP-29 is thought to be a negative modulatorof neurotransmitter release and a key component in intracellular proteintrafficking pathways, with mutations to SNAP-29 resulting in theneurocutaneous syndrome termed CEDNIK.

Blocking secretion by modulating the activity of SNARE proteins has beendemonstrated by blocking release of neurotransmitters from motorneurons, using botulinum neurotoxins (BoNTs) to degrade neuronal SNAREproteins that mediate neurotransmitter release. Botulinum neurotoxins(BoNTs), a family of zinc endopeptidases produced by the bacteriaClostridium botulinum, are a powerful class of drugs that areFDA-approved for a wide range of therapeutic and cosmetic applications.There are seven widely recognized BoNT serotypes (BoNT/A through G) anda recently reported serotype H. BoNTs cleave one or more solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)proteins found in motor neurons, blocking neurotransmitter release andleading to flaccid paralysis.

Although among the deadliest natural substances known, BoNTs are widelyused in various pharmaceutical and cosmetic applications includingcervical dystonia, hyperhidrosis, strabismus, blepharospasm, glabellarlines, and chronic migraine. During intoxication, BoNTs selectively bindto and enter motor neurons via the H chain portion of the molecule. Uponentry into the motor neuron the L chain portion of the molecule isreleased and degrades the targeted SNARE protein required for controlledneurotransmitter secretion in a highly sequence-specific manner. Thisresults in specific and long-term reduction in the contraction ofmuscles associated with treated motor neuron. Both binding to motorneurons and degradation of SNAREs utilized in neurotransmitter releaseare highly specific. For example, BoNT/A, the basis of most BoNT-basedpharmaceuticals, blocks secretion from exposed motor neurons byspecifically cleaving the protein SNAP-25 but does not bind to othercell types or cleave other SNAP-25 isoforms (such as those expressed innon-neuronal cells). BoNTs have previously been retargeted tonon-neuronal cell types through H chain modification. However,therapeutic utility of re-targeted BoNTs is limited by proteolyticspecificity for neuronal SNARE proteins. Thus, the therapeutic use ofBoNTs are currently limited to neuron-related diseases/conditions and isineffective for treating non-neuronal secretion disorders.

Thus, there remains a need for BoNTs and/or modified BONTs that exhibittherapeutic secretory inhibition effects in non-neuronal cells.

SUMMARY OF THE INVENTION

The inventive subject matter provides apparatus, systems and methods inwhich a—

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict exemplary results of a method of the inventiveconcept. FIG. 1A depicts results of electrophoresis of Strep-Tactinpurification of an exemplary botulinum LC, along with results from a setof molecular weight standards. FIG. 1B shows typical results obtained ofFRET assays applied to serial dilutions of LC preparations obtained fromeither 5 mL or 200 μL culture volumes of transfected cells, using a FRETconstruct with an LC-cleavable region joining a donor fluorophore to anacceptor fluorophore.

FIG. 2 schematically depicts assay methodologies of the inventiveconcept. Three different entry points for botulinum light chain (LC)into the workflow are shown, representing three different assaymethodologies.

FIG. 3 illustrates alignment of two non-neuronal SNAREs (SNAP-23 andSNAP-29) with a portion of the neuronal SNARE SNAP-25 sequence asrepresented in an exemplary reporting construct directed towardsbotulinum neurotoxin A or E. The α-exosite and β-exosite recognitionregions of the SNAP-25 sequence are indicated, with residues thatinteract with specified amino acids of the light chain (LC) of botulinumneurotoxin A indicated by arrows. The SNAP-25 cleavage site associatedwith botulinum neurotoxin A LC activity is also shown.

FIG. 4 shows an exemplary microwell plate where the arrangement of testsperformed in individual wells facilitates high throughput testing usingmethods of the inventive concept.

DETAILED DESCRIPTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

The inventive subject matter provides compositions and methods forproducing and identifying compositions that provide a mutated BoNT lightchain (LC) that has proteolytic activity with non-neuronal SNAREproteins. Non-neuronal SNARE proteins encompass SNARE proteins involvedin secretory processes of non-neuronal cells, including neuroendocrinecells. Selected amino acids within the light chains (LCs) of extant BoNTproteins, such as BoNT/A, can be mutated at one or more sites to providerecognition, substrate specificity, and/or enhanced reaction kineticsfor one or more non-neuronal SNARE protein(s), such as SNAP-23 and/orSNAP-29. LC isolation and characterization methodologies thatidentification of useful or suitable mutated LCs are provided, as aretreatment methodologies utilizing such constructs.

One should appreciate that the disclosed compositions and methodsprovide many advantageous technical effects including provision ofspecific and long-lasting control of hyper-secretion from non-neuronalcells and relief from associated disease.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints, andopen-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary. The recitation of ranges of values herein is merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range. Unless otherwise indicatedherein, each individual value with a range is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

In embodiments of the inventive concept, modified BoNTs with specificityfor non-neuronal SNAREs are derived from native sequences associatedwith Clostridium botulinum neurotoxins, including BoNT/A, BoNT/B,BoNT/C, BoNT/D, BoNT/E and BoNT/F. These neurotoxins include light chain(LC) portions, for example the light chain of BoNT/A (SEQ ID NO 4) thatspecifically bind to and exhibit proteolytic activity against neuronalSNAREs. Embodiments of the inventive concept include peptides derived bysite-specific mutation of one or more selected amino acids within an LCsequence, as detailed below.

Such mutations can be provided in the form of bacteria, yeast, and/orother cells carrying expression vectors encoding for a peptide ofinterest. Such expression vectors can be the result of transientinfection, and can be inducible or noninducible. The presence of amutated BoNT LC with enhanced binding to and/or substrate specificityfor a non-neuronal SNARE can be identified using an in vitro assay,which lends itself to automation.

BoTest® reporters are used widely used in high throughput screening(HTS) studies to identify BoNT inhibitors and in BoNT-based drug productpotency testing. Commercial BoTest® assays utilize a fusion peptidereporter that includes a Førster energy resonance transfer (FRET) pairof peptide fluorophores separated by a portion of a neuronal SNAREprotein substrate. Proteolysis of the neuronal SNARE protein substrateresults in a separation of the FRET pair, resulting in a change in theobservable fluorescent that permits sensitive and accurate quantitativemeasurement of BoNT proteolytic cleavage. In order to characterizemutated BoNT peptides with the desired non-neuronal SNARE specificity,modified BoTest reporters are provided that incorporate non-neuronalSNARE sequences representative of the desired SNARE protein specificityinterposed between the FRET pair of peptide fluorophores. For example,to characterize mutated BoNTs with enhanced binding to and/or substratespecificity for SNAP-23, a reporter construct incorporating all or aportion of the SNAP-23 amino acid sequence (for example, SEQ ID NO 2)interposed between a FRET pair of fluorescent peptides (for example,yellow fluorescent protein and cyan fluorescent protein) can beutilized. Similarly, to facilitate identification of mutated BoNTs withenhanced binding to and/or substrate specificity for SNAP-29, a reporterconstruct incorporating all or a portion of the SNAP-29 amino acidsequence (for example, SEQ ID NO 3) interposed between a FRET pair offluorescent peptides (for example, yellow fluorescent protein and cyanfluorescent protein) can be utilized.

In some embodiments, a reporting construct can include an anchoringregion. Such an anchoring region can serve to localize the reportingconstruct to a test surface or membrane. In such embodiments thecleavage site is interposed between the anchoring region and a reportingregion (for example, one or more fluorescent proteins). Cleavage of thecleavage site results in release of the reporter from the test surfaceor from the membrane. This cleavage activity can be detected in anynumber of ways, including characterization of residual signal from thereporter at the test surface or membrane region, detection of signalfrom the reporter following release, or loss of signal from the reporterdue to degradation of the reporter region following release from thetest surface or membrane. In a preferred embodiment, the anchoringregion provides localization to a lipid membrane. Suitable lipidmembranes include a cell membrane, plasma membrane, vesicle membrane,and/or a lipid layer applied to or supported by a surface. In someembodiments of the inventive concept, the reporting construct can beboth expressed and mutated BoNT activity characterized within the sameliving cell.

As noted above, BoNTs occur in a number of different serotypes: BoNT/A,BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, and BoNT/G. A BoNT/H has alsobeen recently proposed. These differ in amino acid sequence, duration ofaction, and/or substrate specificity. For example both BoNT/A and BoNT/Ehave substrate specificity for SNAP-25 (SEQ ID NO 1), however BoNT/A hasa duration of action of some months whereas BoNT/E has a duration ofaction of a few days. BoNTs utilized for mutation to alter substratespecificity can be selected, at least in part, on the basis of a desiredduration of action. In some embodiments, only the light chain (LC)sequence (i.e. the portion of the BoNT that provides substraterecognition and proteolytic activity) of the selected BoNT is mutated.In a preferred embodiment the LC peptide of BoNT/A (LC/A, SEQ ID NO 4)serves as the basis of the mutated BoNT peptide.

One embodiment of the inventive concept is a method for identifyingmutated BoNT peptides that have improved substrate specificity and/orreaction kinetics for a non-neuronal SNARE when compared to acorresponding peptide (i.e. without the mutation(s)) having a nativeBoNT sequence. Such a mutated BoNT peptide can, for example, demonstratea binding energy for a non-neuronal SNARE that is 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, or greater than that of a correspondingpeptide having a native BoNT sequence. Similarly, such a mutated BoNTpeptide can show reaction kinetics indicative of proteolytic cleavage ofa target non-neuronal SNARE that is 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or greater than that of a corresponding peptide having anative BoNT sequence. In some embodiments, screening studies utilizingmodified BoTest reporting constructs, as described above, can beperformed on full length or truncated LC sequences to identify thosewith a desirable characteristic. Use of full-length sequencesadvantageously provides a more accurate representation of the intactBoNT and supports purification protocols that provide highly purifiedand active peptide products. In a preferred embodiment, screeningstudies are performed on full length (448 amino acids) LC/A (SEQ ID NO4) or LC/A derived sequences. Miniaturized, simplified, and re-optimizedprotocols for culture volumes ranging from 5 ml to 200 μl cultures canbe realized through the use of Strep-Tactin® spin columns.

In an example of a typical screening study, 5 ml and 200 μl culturesproduced assayable quantities of LC/A. Using LC/A with the nativesequence, complete BoTest® A/E reporter cleavage is typically observedin less than 1 hour with high dilutions (for example, 1:20,000) of apreparation from a 200 μl sample. Extended incubation can improvesensitivity. For example, extending the incubation time to 18 hourstypically demonstrates detectable activity from a 1:200,000 dilution ofa typical 200 μl-scale preparation of native sequence LC/A. In someembodiments of the inventive concept, a control reporting construct isprovided in which the donor and acceptor fluorophores are separated by acleavage site that is not represented on either neuronal or non-neuronalSNAREs. Such a control reporting construct is useful for determinationof nonspecific protease activity, for example due to contamination orundesirable activity on the part of the mutated LC. An example of such acontrol reporting construct is BoTest® KO, a BoNT-insensitive controlversion of the BoTest® A/E reporting construct with sensitivity tonon-BoNT proteases.

Examples of identification of BoNT LC/A mutations that can recognize thenon-neuronal SNARE proteins SNAP-23 and SNAP-29 follow.

LC/A can be obtained from as little as 200 μl of bacterial culture,allowing protein expression in a single well of a 96-well microwellplate (see FIG. 1). As shown, reduction of the culture volume from theconventional 5 mL volume to 200 μL (which can be convenientlyaccommodated within a single well of a conventional 96 well microwellplate) has no apparent impact on the yield or activity of an LC derivedfrom a transformed cell. Commercially available Strep-Tactin®-coated96-well plates (from IBA) can be used to perform some or all assay stepsin a single well (FIG. 2), as the expressed mutated LC/A binds to thewell surface following bacterial lysis and can be purified by washingbefore assaying. The assay can subsequently be performed by adding areaction buffer containing a SNAP-23 or SNAP-29 containing reportingconstruct to the wells and incubating. This method provides high assaythroughput and reduced time, cost, and sample manipulation.

As shown in FIG. 2, there are several useful testing strategies.Workflow is similar in all of these testing strategies—culture andinduction, followed by lysis of the induced cells and isolation of theexpressed LC, followed by characterization of the activity of theexpressed LC. The methods differ in the point of entry of the LC intothe testing process. In some embodiments, growth and induction, lysisand isolation, and activity testing can occur in the same test well. Inother embodiments growth, induction, and lysis is performed in anothervessel or test fixture, while isolation of the LC and characterizationof its activity take place in the same well of a test plate. In stillanother embodiment, growth, induction, lysis, and isolation of the LCtake place in a vessel(s) and/or a fixture(s) that is separate from thetest plate, and the isolated LC is transferred to a well of the testplate for characterization of its activity. In some embodiments, asingle test plate may be used to carry out two or more of these testingworkflows simultaneously. As shown in FIG. 2, in some instancesStrep-Tactin®-coated plates can be used for the entire assay workflow,in others such plates are used only for protein purification and/orscreening tests.

As shown in FIG. 2, mutated LC/A-expressing bacteria can be cultured ina conventional 96-well microwell plate and then inoculated into freshmedium in either standard (Strategies 2 and 3 of FIG. 2) orStrep-Tactin®-coated (Strategy 1 of FIG. 2) 96-well plates where theyare cultured, induced, and incubated for a period of time sufficient forgrowth and expression of the mutated LC/A (for example, overnight).Following induction, the bacteria are lysed. Expressed mutated LC/A fromthe Strep-Tactin®-coated plate cultures binds to the walls of the platewell, while lysed cultures grown in standard plates can be added toStrep-Tactin®-coated plates and tested. Test wells can be subsequentlywashed with suitable wash buffer (for example phosphate-buffered salinecontaining 0.1% Tween-20 (PBS-T)) to remove unbound materials. Lysedcultures can also be purified using Strep-Tactin® spin columns andtested in the wells of conventional microwell plates. In someembodiments of the inventive concept a control reporting construct, suchas BoTest® KO, is added to at least some test wells to monitornon-specific protease activity.

Since mutants with altered substrate specificity might have alteredreaction rates and/or catalytic turnover relative to native LC/Acleavage of SNAP-25 (SEQ ID NO 1), assay detection limits can bedetermined by titrating the amount of induced bacterial culture usedduring purification and assaying to determine the dilution that gives aresponse that differs by about 2, 3, 4, 5, 6, 7, 8, 9, 10 or morestandard deviations from that of a control containing no inducedculture. In a preferred embodiment, mutated LCs tested in such a fashionproduce detectable cleavage of an appropriate detecting peptideconstruct at a dilution of at least about 1:100 within at least about 4hours.

SNAP-23 and SNAP-29 reporting peptide constructs can be generated bysubstituting all or portions of the SNAP-23 and/or SNAP-29 sequence fora corresponding portion of a previously characterized BoNT reportingconstruct. For example, a SNAP-23 or SNAP-29 reporting peptide constructcan be produced by exchanging the SNAP-25 fragment in BoTest® A/E withportions of or full length SNAP-23 (for example, SEQ ID NO 2) andSNAP-29 (for example, SEQ ID NO 3), respectively. Since nominal SNAP-23and SNAP-29 insertion fragments (SEQ ID NOs 2 and 3, respectively) areslightly shorter than SNAP-25 (SEQ ID NO 1) (FIG. 3); spacer peptidesequences can be added to maintain size consistency across allreporters. Expression vectors can be constructed, expressed, and theresulting reporting peptides purified, for example for in vitro testingpurposes. The size, purity, and yield of the reporting constructs thusproduced can be quantified. SNAP-23 and SNAP-29 reporting constructs canbe tested with BoNT/A and/or BoNT/E to verify lack of substrate activitywith these proteases. Cleavage of such constructs by non-BoNT proteases(for example, trypsin) can be used to verify fluorescent peptideperformance.

The sites of binding and/or recognition interactions between the BoNT/Alight chain (LC) and a portion of SNAP-25 (SEQ ID NO 1) are shown inFIG. 3, along with sequence alignment between SNAP-25 (SEQ ID NO 1) andthe non-neuronal SNAREs SNAP-23 (SEQ ID NO 2) and SNAP-29 (SEQ ID NO 3).Specific amino acids involved in the interaction and the correspondinginteracting amino acids of the BoNT/A LC are indicated by arrows. Thesite on SNAP-25 (SEQ ID NO 1) that is cleaved by the BoNT A LC is alsoshown. The α- and β-exosites indicated represent regions of SNAP-25 (SEQID NO 1) that, when occupied, inhibit the reaction with the BoNT/A LC.

Development of modified LC/As with the desired, altered substratespecificity can be accomplished using site-directed LC/A mutagenesisthat target critical residues identified as relevant to LC/A:SNAP-25interaction, thereby generating libraries of mutated LC peptides. Suchlibraries can be screened for novel substrate specificity and/orreaction kinetics as described above.

Unlike many proteases, BoNTs require large substrates for optimalcleavage. The geometries and compositions of LC active sites are highlyconserved across BoNT serotypes, suggesting that substrate specificityarises from LC:substrate binding that first occurs via exositeinteractions that orient the substrate, stabilize the complex, andpromote additional contacts that poise the LC for cleaving activity. Theinventors have realized that cleavage of non-native SNARE isoforms is afunction of effective substrate binding, as LC active sites are highlyconserved, are not involved in substrate side chain interactions, andthe reaction involved in cleavage of the peptide chain does not requirethe presence of specific amino acid side chains at the cleavage site.

For example, the LC/A contact residues responsible for SNAP-25 (SEQ IDNO 1) interaction are highly or partially conserved in the correspondingresidues of SNAP-23 and SNAP-29 (see FIG. 3). A mix of saturation andspecific mutagenesis can be used to generate different categories ofmutants based on the SNAP domain that they align with, such as theα-exosite, extended linker, and active site residues. BoNT/A LChydrophobic side-chain interactions that extend along the SNAP-25α-exosite are largely conserved in SNAP-23 and SNAP-29 (see FIG. 3).However, both SNAP-23 and SNAP-29 contain substitutions at SNAP-25Asp166, disrupting a salt bridge with LC/A Lys337 and possibly changingbinding stability. SNAP-29 also contains a SNAP-25 Gln152 substitution,which potentially affects a polar side chain contact with LC/A Lys356,and an additional non-conservative Gly substitution at SNAP-25 Ile156.Saturation mutagenesis at LC/A Lys356 and Lys337 can be used to generateLC/A mutants that compensate for the loss of the stabilizing salt bridgeand polar side-chain contacts on interaction with SNAP-23 and SNAP-29.

A salt bridge between Arg176 of SNAP-25 and Glu148 of LC/A provides apotentially critical anchor point for substrate positioning and that islost with both SNAP-23 and SNAP-29. Mutant LC/A peptide libraries thatreestablish that salt bridge in SNAP-29 can be provided by mutating LC/AGlu148 to Lys or Arg. SNAP-23 contains a Pro substitution at thisposition, so alternate salt bridges for mutant LC/A directed to thissubstrate can be provided by mutating Val304 and Ser143 of LC/A to Aspor Glu to exploit the neighboring Lys and Arg residues. A polarside-chain interaction occurs between SNAP-25 (SEQ ID NO 1) Glu183 andLC/A Asn136 and is disrupted in both SNAP-23 and SNAP-29. In SNAP-23,mutant LC/A libraries that compensate for this can be provided bygenerating a salt bridge by mutating LC/A Asn136 to either Lys or Arg.Similarly, mutant LC/A libraries can also be generated by saturationmutation at this position to screen for mutants that compensate for theThr substitution in SNAP-29.

The active pockets of SNAP-23 and SNAP-29 include large, positivelycharged residues (Lys and Arg, respectively) in place of the SNAP-25Thr200. These substitutions can be accommodated by enlarging the pocketand/or introducing a negatively charged residue at Leu256 and Val258 inLC/A. Asp, Ala, and Gly substitutions can be made at these positions toenlarge and/or make the active pocket more favorable for SNAP-23 and/orSNAP-29. SNAP-29 also contains two significant Lys and Glu substitutionsat SNAP-25 Asp193 and Asn196, respectively, that interact with LC/AThr176 and His227 and result in inverting or introducing chargedside-groups. Saturated libraries at LC/A Thr176 and His227 can beconstructed to screen for mutants that can accommodate these changes inSNAP-29.

Examples of mutations of the BoNT/A light chain that can be consideredsuitable for mutated LCs of the inventive concept are summarized inTable 1, which indicates substitutions at specified sites within thesequence of the botulinum serotype A neurotoxin light chain with an “X”.It should be appreciated a mutated LC of the inventive concept caninclude a single substitution and can also include two or more of thesesubstitutions.

TABLE 1 Substitution Asn136 Ser143 Glu148 Val304 Thr176 His227 Lys337Leu256 Val258 Lys356 Ala SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID NO 5 NO 30 NO 49 NO 68 NO 87 NO 90 NO 93 Arg SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID NO 6 NO 26 NO 31 NO 50 NO 69 NO 94 Asn SEQ ID SEQ IDSEQ ID SEQ ID NO 32 NO 51 NO 70 NO 95 Asp SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 7 NO 24 NO 28 NO 33 NO 52 NO 71 NO88 NO 91 NO 96 Cys SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 8 NO 34 NO 53NO 72 NO 97 Gln SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 9 NO 35 NO 54 NO73 NO 98 Glu SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 10 NO25 NO 29 NO 36 NO 55 NO 74 NO 99 Gly SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID NO 11 NO 37 NO 56 NO 75 NO 89 NO 92 NO 100 His SEQ ID SEQID SEQ ID SEQ ID NO 12 NO 38 NO 76 NO 101 Ile SEQ ID SEQ ID SEQ ID SEQID SEQ ID NO 13 NO 39 NO 57 NO 77 NO 102 Leu SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID NO 14 NO 40 NO 58 NO 78 NO 103 Lys SEQ ID SEQ ID SEQ ID SEQ ID NO15 NO 27 NO 41 NO 59 Met SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 16 NO 42NO 60 NO 79 NO 104 Phe SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 17 NO 43 NO61 NO 80 NO 105 Pro SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 18 NO 44 NO 62NO 81 NO 106 Ser SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 19 NO 45 NO 63 NO82 NO 107 Thr SEQ ID SEQ ID SEQ ID SEQ ID NO 20 NO 64 NO 83 NO 108 TrpSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 21 NO 46 NO 65 NO 84 NO 109 TyrSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 22 NO 47 NO 66 NO 85 NO 110 ValSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 23 NO 48 NO 67 NO 86 NO 111In other embodiments of the inventive concept, one or more of Asn136,Ser143, Glu148, Val304, Thr176, His227, Lys337, Leu256, Va1258, and/orLys356 can be substituted with any non-corresponding amino acid withinLC/A (SEQ ID NO 4) to provide all or part of a protease with enhancedaffinity and/or reaction kinetics for a non-neuronal SNARE proteinrelative to native LC/A.

LC/A mutations with substrate specificity for SNAP-23 and/or SNAP-29 canbe identified by generating the appropriate mutation collections andtesting against SNAP-23 and SNAP-29 reporting constructs. In a typicalexample of a testing protocol, a 96-well microwell plate can accommodate20 mutant LC/A peptides plus controls, allowing the rapid processing ofpeptide library contents. Single colonies from the mutant LC librarycolonies can be cultured and assayed. The original colonies can bestored at 4° C. during testing to permit clone recovery and/orretesting. Generally, plates can be evaluated only if a positive (i.e.wild-type) LC/A control shows full cleavage of BoTest® A/E and nosignificant (i.e. <10%) negative control reporting construct (forexample, BoTest® KO) cleavage after 4 hours of incubation with thesereporting peptide constructs. Plates that initially fail these criteriacan be retested. A positive mutant LC/A can be defined as any mutantLC/A that shows greater than about 20% appropriate reporter cleavage inone or both samples of a duplicate. The original parental culture forany positive result can be cultured, and glycerol stocks generated andbanked. All positive clones can be retested, and clones that retestpositive can be banked and fully sequenced to identify mutations. Clonesthat show positive results can be verified, for example usingrecombinant full-length SNAP-23 or SNAP-29 and protein blot analysis toconfirm substrate cleavage, and can be transfected into cells to measuresecretion reduction in vivo.

The integrated growth/induction, lysis, isolation, and activity assaysdescribed above make identification of such mutant LCs possible. In someembodiments, however, it can be useful to implement high throughput,automated, or semi-automated (i.e. combining manual and automated steps)screening methods to facilitate such identification. In some embodimentsof the inventive concept, such methods can be implemented using a 24,48, 96, 384, and/or 1,536 well plate. In some embodiments all of thesteps of growth, induction, lysis, isolation, and activity testing areperformed on a single plate. In other embodiments these steps can beperformed across two or more plates for a given mutant LC chain beingcharacterized. In still other embodiments, one or more of these stepscan be performed offline (for example, using a small affinity column forisolation of the mutant LC) prior to addition to a well of a microwellplate.

An exemplary arrangement of a 96-well microwell plate that facilitateshigh throughput and/or automated testing of LC mutations for screeningpurposes is shown in FIG. 4. As shown, the plate includes LC/A positiveand empty vector negative controls that are tested using a BoTest A/Ereporting construct and a non-specific protease directed reportingconstruct (for example, BoTest KO). LC mutations can be tested usingreporting constructs carrying suitable non-neuronal SNARE proteins (forexample, SNAP-23 or SNAP-29) or cleavage fragments thereof interposedbetween donor and acceptor fluorophores of a FRET pair. In the platelayout depicted, each LC mutation is tested against such a non-neuronalSNARE reporting construct in duplicate. In addition, every fifth LCmutation is tested against a non-specific protease directed reportingconstruct (such as BoTest KO) in duplicate as a control.

It should be appreciated that light chain sequences identified as havingthe desired activity for non-neuronal SNARE proteins can be combinedwith a targeting moiety that provides selective binding to anon-neuronal cell. Suitable targeting moieties include native or mutatedbotulinum heavy chain sequences which, when combined with an LC, providean intact, functional protein capable of targeting cells, beinginternalized, and being processed. In some embodiments of the inventiveconcept, one or more mutant LC chains are combined with one or morenative or mutated heavy chains of BoNT/A, BoNT/B, BoNT/C, BoNT/D,BoNT/E, BoNT/F and/or BoNT/G. It should be appreciated that a mutant LCderived from a given BoNT serotype can be combined with a nativesequence or mutated heavy chain from a corresponding BoNT serotype or adifferent BoNT serotype. In other embodiments of the inventive concept,one or more mutant LCs are combined with one or more mutated botulinumheavy chains that show altered cellular specificity for a desired celltype.

In other embodiments of the inventive concept, a targeting moiety can bea peptide or other molecule that does not correspond to a botulinumneurotoxin heavy chain. For example, a targeting moiety can be anantibody, and antibody fragment, or a single chain antibody. In otherembodiments a targeting moiety can be a ligand for a cell surfacereceptor (for example, a drug, hormone, saccharide, polysaccharide, orlipopolysaccharide), or a receptor for a ligand existing on the surfaceof the target cell (for example, a lectin). In other embodiments, thetargeting moiety can be one member of an affinity pair (for example,biotin and avidin), with the other member of the affinity pair having(or being part of a molecule that has) an affinity for the target cell.In still other embodiments, a non-peptide macromolecule (such as anaptamer) can be used as a targeting moiety. In some embodiments, atargeting moiety can be joined to a mutant LC by a linker peptide. Suchlinker peptides can be selected to be flexible (for example, to reducesteric hindrance) or can be selected to be rigid (for example, toprovide a desired geometry). In some embodiments the linker peptide isselected to be cleaved or degraded by cellular processes followinginternalization, thereby releasing the mutant LC.

Another embodiment of the inventive concept is a method for treating anindividual with a disease characterized by hypersecretion. In such amethod, a drug composition that includes a mutant LC targeting anon-neuronal SNARE is administered to the patient having such a diseaseor in need of such treatment. In such an embodiment, the mutant LC canbe selected to have substrate specificity and/or enhanced reactionkinetics (relative to the native sequence LC from which it is derived)for one or more non-neuronal SNARE proteins associated with secretion,where such a SNARE protein(s) is found in a cell characterized byhypersecretion in the afflicted individual. Such a drug composition canbe in the form of an injectable liquid, and in such form can includebuffers and preservatives suitable for intravenous, intramuscular,subdermal, intraocular, peritoneal, and/or central nervous system usage.In other embodiments the drug composition can be supplied as a topicalpreparation, such as a suspension, ointment, gel, lotion, or cream. Insuch embodiments the drug composition can include additionalingredients, such as emollients, excipients, and/or agents that aid intransdermal delivery. In some of such embodiments, the drug compositioncan be supplied in the form of a patch or film that is appliedtopically. In still other embodiments, the drug composition can besupplied in a form that facilitates transmucosal delivery. In suchembodiments the drug composition can be supplied as an inhaled substance(for example, a powder, vapor, and/or droplet mist), a sublingual dropor lozenge, a nasal spray, an eye drop, suppository, or pessary. Instill other embodiments the drug composition can be supplied for oralconsumption, for example as a consumable liquid or food. In suchembodiments the drug composition can include colorants, flavorants,and/or thickening agents. When packaged for oral administration, a drugcomposition including a mutant LC can include formulations or mechanismsto delay release and/or absorption until a desired location with thegastrointestinal tract is reached (for example, time release coatings,capsules with perforations, etc.).

Treatment of a hypersecretion disease using a modified LC light chaincan be performed using a dosing/schedule that permits effectivetreatment while minimizing undesired effects. For example, a mutant LCof the inventive concept can be administered at dosages ranging from 1mg/kg body weight to 100 mg/kg body weight. In some embodiments of theinventive concept, a single dose of a drug composition including amutant LC of the inventive concept can be sufficient to derive abeneficial effect. In other embodiments of the inventive conceptmultiple doses over a period of time are administered. For example,relatively small doses of a drug composition including the mutant LC canbe administered on a regular schedule (e.g. every other day, daily, twoto 12 times a day) until the desired result is achieved. Due to theirmethod of action a drug composition containing a mutant LC can have anextended effect once the desired therapeutic effect is achieved. Asnoted above, the duration of this effect can be selected based, at leastin part, on the selection of the native LC sequence from which themutant LC is derived. In some embodiments, therapeutic effects maypersist for at least 3 months following administration of the mutant LC.In other embodiments, therapeutic effects may persist for a day, 3 days,a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, or more followingadministration of an effective dose of the mutant LC.

In some embodiments of the inventive concept, a series of mutant LCshaving similar substrate specificities but different sequences can beadministered to reduce the effect of patient antibodies developed to amutant LC and/or to reduce the induction of such an antibody response ina patient treated with such compositions. In still other embodiments, amixture of mutant LCs having similar substrate specificities butdifferent sequences can be administered, thereby reducing theconcentration of each mutant LC below a level likely to induce asignificant immune response while maintaining a therapeutic effect.Similarly, mutant LC can be modified to reduce antigenicity (forexample, via PEGylation).

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refer to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A protease with substrate specificity for anon-neuronal SNARE protein, comprising a mutated botulinum toxin lightchain having a non-native exosite recognition sequence, wherein thenon-native exosite recognition sequence comprises a peptide sequencehaving a mutation relative to a peptide sequence corresponding to anative botulinum toxin exosite sequence, and wherein the mutation isselected to provide improved binding between the mutated botulinum toxinlight chain to a non-neuronal SNARE protein than to a neuronal SNAREprotein that acts as a substrate for a native botulinum toxin lightchain having the corresponding native botulinum toxin exosite sequence.2. The protease of claim 1, wherein the mutated botulinum toxin lightchain is derived from a botulinum toxin sequence corresponding to alight chain sequence of at least one of the group consisting of BoNT/A,BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, and BoNT/G.
 3. The protease ofclaim 1, wherein the non-native exosite recognition sequence comprisesone or more mutations to sites corresponding to one or more of the groupconsisting of Asn136, Ser143, Glu148, Val304, Thr176, His227, Lys337,Leu256, Val258, and Lys356 of SEQ ID NO.
 4. 4. The protease of claim 3,wherein the mutation of Asn 136 is selected from the group consisting ofAla, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, and Val.
 5. The protease of claim 3, wherein themutation of Ser143 is selected from the group consisting of Asp and Glu.6. The protease of claim 3, wherein the mutation of Glu148 is selectedfrom the group consisting of Lys and Arg.
 7. The protease of claim 3,wherein the mutation of Val304 is selected from the group consisting ofAsp and Glu.
 8. The protease of claim 3, wherein the mutation of Thr176is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu,Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, and Val. 9.The protease of claim 3, wherein the mutation of His 227 is selectedfrom the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
 10. Theprotease of claim 3, wherein the mutation of Lys337 is selected from thegroup consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile,Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
 11. The protease ofclaim 3, wherein the mutation of Leu256 is selected from the groupconsisting of Asp, Ala, and Gly.
 12. The protease of claim 3, whereinthe mutation of Val258 is selected from the group consisting of Asp,Ala, and Gly.
 13. The protease of claim 3, wherein the mutation ofLys356 is selected from the group consisting of Ala, Arg, Asn, Asp, Cys,Glu, Gln, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, andVal.
 14. The protease of claim 1, wherein the non-neuronal SNARE proteinis selected from the group consisting of SNAP-23 and SNAP-29.